Hydrogel tissue adhesive for medical use

ABSTRACT

A hydrogel tissue adhesive formed by reacting an aldehyde-functionalized polysaccharide containing pendant aldehyde groups with a water-dispersible, multi-arm amine is described. The hydrogel may be useful as a tissue adhesive or sealant for medical applications that require a more rapid degradation time, such as the prevention of undesired tissue-to tissue adhesions resulting from trauma or surgery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/379,843, Filed Dec. 21, 2011 and U.S. ProvisionalApplication Ser. Nos. 61/222,713 and 61/222,720, both filed on Jul. 2,2009. The contents of each of these priority applications areincorporated herein by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The invention relates to the field of medical adhesives. Morespecifically, the invention relates to a hydrogel tissue adhesive formedby reacting an aldehyde-functionalized polysaccharide containing pendantaldehyde groups with a water-dispersible, multi-arm amine.

BACKGROUND OF THE INVENTION

Tissue adhesives have many potential medical applications, includingwound closure, supplementing or replacing sutures or staples in internalsurgical procedures, preventing leakage of fluids such as blood, bile,gastrointestinal fluid and cerebrospinal fluid, adhesion of syntheticonlays or inlays to the cornea, drug delivery devices, and asanti-adhesion barriers to prevent post-surgical adhesions. Conventionaltissue adhesives are generally not suitable for a wide range of adhesiveapplications. For example, cyanoacrylate-based adhesives have been usedfor topical wound closure, but the release of toxic degradation productslimits their use for internal applications. Fibrin-based adhesives areslow curing, have poor mechanical strength, and pose a risk of viralinfection. Additionally, fibrin-based adhesives do not bond covalentlyto the underlying tissue.

Several types of hydrogel tissue adhesives have been developed, whichhave improved adhesive and cohesive properties and are nontoxic (see forexample Sehl et al., U.S. Patent Application Publication No.2003/0119985, and Goldmann, U.S. Patent Application Publication No.2005/0002893). These hydrogels are generally formed by reacting acomponent having nucleophilic groups with a component havingelectrophilic groups, which are capable of reacting with thenucleophilic groups of the first component, to form a crosslinkednetwork via covalent bonding. However, these hydrogels typically swell,dissolve away too quickly, or lack sufficient adhesion or mechanicalstrength, thereby decreasing their effectiveness as surgical adhesives.

Kodokian et al. (copending and commonly owned U.S. Patent ApplicationPublication No. 2006/0078536) describe a polysaccharide-based hydrogeltissue adhesives formed by reacting an oxidized polysaccharide with awater-dispersible, multi-arm polyether amine. These adhesives provideimproved adhesion and cohesion properties, crosslink readily at bodytemperature, maintain dimensional stability initially, do not degraderapidly, and are nontoxic to cells and non-inflammatory to tissue.However, for certain applications, such as the prevention of undesiredtissue-to tissue-adhesions resulting from trauma or surgery, a morerapidly degrading hydrogel tissue adhesive is needed. For example, anadhesion prevention composition should not persist at the site once thehealing process has begun, typically not longer than 1 to 3 weeks.

Therefore, the need exists for a hydrogel tissue adhesive that has thedesirable properties of the oxidized polysaccharide-based tissueadhesives described by Kodokian et al., supra, but has a shorterdegradation time.

SUMMARY OF THE INVENTION

The present invention addresses the above need by providing a hydrogeltissue adhesive that has good adhesion and cohesion properties,crosslinks readily at body temperature, maintains dimensional stabilityinitially, is nontoxic to cells and non-inflammatory to tissue, anddegrades more rapidly than the oxidized polysaccharide-based hydrogeltissue adhesives.

Accordingly, in one embodiment the invention provides a kit comprising:

-   -   a) at least one aldehyde-functionalized polysaccharide        containing pendant aldehyde groups, said aldehyde-functionalized        polysaccharide having a weight-average molecular weight of about        1,000 to about 1,000,000 Daltons and a degree of aldehyde        substitution of about 10% to about 200%; and    -   b) at least one water-dispersible, multi-arm amine wherein at        least three of the arms are terminated by at least one primary        amine group, said multi-arm amine having a number-average        molecular weight of about 450 to about 200,000 Daltons.

In another embodiment, the invention provides a dried hydrogel formed bya process comprising the steps of:

-   -   a) combining in a solvent (i) at least one        aldehyde-functionalized polysaccharide containing pendant        aldehyde groups, said aldehyde-functionalized polysaccharide        having a weight-average molecular weight of about 1,000 to about        1,000,000 Daltons and a degree of aldehyde substitution of about        10% to about 200% with (ii) at least one water-dispersible,        multi-arm amine, wherein at least three of the arms are        terminated by at least one primary amine group, said multi-arm        amine having a number-average molecular weight of about 450 to        about 200,000 Daltons, to form a hydrogel; and    -   b) treating said hydrogel to remove at least a portion of said        solvent to form the dried hydrogel.

In another embodiment, the invention provides a composition comprisingthe reaction product of:

-   -   a) at least one aldehyde-functionalized polysaccharide        containing pendant aldehyde groups, said aldehyde-functionalized        polysaccharide having a weight-average molecular weight of about        1,000 to about 1,000,000 Daltons and a degree of aldehyde        substitution of about 10% to about 200%, and    -   b) at least one water-dispersible, multi-arm amine wherein at        least three of the arms are terminated by at least one primary        amine group, said multi-arm amine having a number-average        molecular weight of about 450 to about 200,000 Daltons.

In another embodiment, the invention provides a crosslinked hydrogelcomposition comprising:

-   -   a) at least one aldehyde-functionalized polysaccharide        containing pendant aldehyde groups, said aldehyde-functionalized        polysaccharide having a weight-average molecular weight of about        1,000 to about 1,000,000 Daltons and having a degree of aldehyde        substitution of about 10% to about 200%; and    -   b) at least one water-dispersible, multi-arm amine wherein at        least three of the arms are terminated by at least one primary        amine group, said multi-arm amine having a number-average        molecular weight of about 450 to about 200,000 Daltons;        wherein said at least one aldehyde-functionalized polysaccharide        and said at least one water-dispersible, multi-arm amine are        crosslinked through covalent bonds formed between the pendant        aldehyde groups of the polysaccharide and the primary amine        groups of the water-dispersible, multi-arm amine.

DETAILED DESCRIPTION

As used above and throughout the description of the invention, thefollowing terms, unless otherwise indicated, shall be defined asfollows:

The term “aldehyde-functionalized polysaccharide” as used herein, refersto a polysaccharide that has been chemically modified to introducependant aldehyde groups into the molecule. The pendant aldehyde groupsmay be single aldehyde groups or dialdehydes. As defined herein,aldehyde-functionalized polysaccharides do not include polysaccharidesthat are oxidized by cleavage of the polysaccharide rings to introducealdehyde groups. Oxidation of the polysaccharide rings results indialdehydes formed by opening the rings of the polysaccharide.

The term “pendant aldehyde group” refers to an aldehyde group that isattached to the carbohydrate of the polysaccharide via one of the ringhydroxyl groups.

The term “degree of aldehyde substitution” refers to the mole percent ofpendant aldehyde groups per mole of carbohydrate repeat units, i.e.,(moles of pendant aldehyde groups/moles of carbohydrate repeatunits)×100.

The term “water-dispersible, multi-arm amine” refers to a polymer havingthree or more polymer chains (“arms”), which may be linear or branched,emanating from a central structure, which may be a single atom, a coremolecule, or a polymer backbone, wherein at least three of the branches(“arms”) are terminated by at least one primary amine group. Thewater-dispersible, multi-arm amine is water soluble or is able to bedispersed in water to form a colloidal suspension capable of reactingwith a second reactant in aqueous solution or dispersion.

The term “dispersion” as used herein, refers to a colloidal suspensioncapable of reacting with a second reactant in an aqueous medium.

The term “water-dispersible, multi-arm polyether amine” refers to awater-dispersible, multi-arm amine wherein the polymer is a polyether.

The term “polyether” refers to a polymer having the repeat unit [—O—R]—,wherein R is a hydrocarbylene group having 2 to 5 carbon atoms. Thepolyether may also be a random or block copolymer comprising differentrepeat units which contain different R groups.

The term “hydrocarbylene group” refers to a divalent group formed byremoving two hydrogen atoms, one from each of two different carbonatoms, from a hydrocarbon.

The term “branched polyether” refers to a polyether having one or morebranch points (“arms”), including star, dendritic, comb, highlybranched, and hyperbranched polyethers. Branches radiate from one ormore trifunctional or higher functional branch points.

The term “dendritic polyether” refers to a highly branched polyetherhaving a branching structure that repeats regularly with each successivegeneration of monomer radiating from a core molecule.

The term “comb polyether” refers to a branched polyether in which linearside-chains emanate from trifunctional branch points on a linear polymerbackbone.

The term “star polyether” refers to a branched polyether in which linearside-chains emanate from a single atom or a core molecule having a pointof symmetry.

The term “hyperbranched polyether” refers to a highly branched polyetherwhich is more branched than “highly branched,” with order approachingthat of an imperfect dendrimer.

The term “highly branched polyether” refers to a branched polyetherhaving many branch points, such that the distance between branch pointsis small relative to the total length of arms.

The term “primary amine” refers to a neutral amino group having two freehydrogens. The amino group may be bound to a primary, secondary ortertiary carbon.

The term “multi-functional amine” refers to a chemical compoundcomprising at least two functional groups, at least one of which is aprimary amine group.

The term “crosslink” refers to a bond or chain of atoms attached betweenand linking two different polymer chains.

The term “crosslink density” is herein defined as the reciprocal of theaverage number of chain atoms between crosslink connection sites.

The term “% by weight”, also referred to herein as “wt %” refers to theweight percent relative to the total weight of the solution ordispersion, unless otherwise specified.

The term “anatomical site” refers to any external or internal part ofthe body of humans or animals.

The term “tissue” refers to any biological tissue, both living and dead,in humans or animals.

The term “hydrogel” refers to a water-swellable polymeric matrix,consisting of a three-dimensional network of macromolecules heldtogether by covalent crosslinks that can absorb a substantial amount ofwater to form an elastic gel.

The term “dried hydrogel” refers to a hydrogel that has been treated toremove at least a portion of the solvent contained therein. Preferably,substantially all of the solvent is removed from the hydrogel.

The term “PEG” as used herein refers to poly(ethylene glycol).

The term “M_(w)” as used herein refers to the weight-average molecularweight.

The term “M_(n)” as used herein refers to the number-average molecularweight.

The term “M_(z)” as used herein refers to the z-average molecularweight.

The term “medical application” refers to medical applications as relatedto humans and animals.

The meaning of abbreviations used is as follows: “min” means minute(s),“h” means hour(s), “sec” means second(s), “d” means day(s), “mL” meansmilliliter(s), “L” means liter(s), “μL” means microliter(s), “cm” meanscentimeter(s), “mm” means millimeter(s), “μm” means micrometer(s), “mol”means mole(s), “mmol” means millimole(s), “g” means gram(s), “mg” meansmilligram(s), “mol %” means mole percent, “Vol” means volume, “w/w”means weight per weight, “Da” means Daltons, “kDa” means kiloDaltons,the designation “10K” means that a polymer molecule possesses anumber-average molecular weight of 10 kiloDaltons, “M” means molarity,“kPa” means kilopascals, “psi” means pounds per square inch, “rpm” meansrevolutions per minute”, “¹H NMR” means proton nuclear magneticresonance spectroscopy, “13-C NMR” means carbon 13 nuclear magneticresonance spectroscopy, “ppm” means parts per million, “cP” meanscentipoise, PBS” means phosphate-buffered saline, “MWCO” means molecularweight cut off.

A reference to “Aldrich” or a reference to “Sigma” means the saidchemical or ingredient was obtained from Sigma-Aldrich, St. Louis, Mo.

Disclosed herein is a hydrogel tissue adhesive formed by reacting analdehyde-functionalized polysaccharide containing pendant aldehydegroups with a water-dispersible, multi-arm amine. The hydrogel may beuseful as a tissue adhesive or sealant for medical applications thatrequire more rapid degradation, including but not limited to, preventionof undesired tissue-to tissue adhesions resulting from trauma orsurgery.

Aldehyde-Functionalized Polysaccharides

Aldehyde-functionalized polysaccharides suitable for use herein arepolysaccharides that have been chemically modified to introduce pendantaldehyde groups into the molecule. The pendant aldehyde groups may besingle aldehyde groups or dialdehydes. The pendant aldehyde groups ofthe aldehyde-functionalized polysaccharides disclosed herein areattached to the polysaccharide through linking groups. In oneembodiment, the linking groups comprise carbon, hydrogen, and oxygenatoms, but do not contain a nitrogen atom, and are attached to thepolysaccharide by ether linkages. As demonstrated in the Examples hereinbelow, aldehyde-functionalized polysaccharides having these types oflinking groups are more stable in aqueous solution that oxidizedpolysaccharides or aldehyde-functionalized polysaccharides having othertypes of linking groups, such as those that contain a nitrogen atom orare linked to the polysaccharide by other chemical linkages (e.g., amideor urethane). In one embodiment, the linking group contains an alkoxygroup alpha to the pendant aldehyde group (i.e., on an adjacent carbonatom). In another embodiment, the linking group does not contain analkoxy group beta to the pendant aldehyde group (i.e., on the secondcarbon atom from the aldehyde group).

As used herein, aldehyde-functionalized polysaccharides do not includepolysaccharides that are oxidized by cleavage of the polysacchariderings to introduce aldehyde groups. Oxidation of the polysacchariderings results in dialdehydes formed by opening the rings of thepolysaccharide. Therefore, the dialdehyde groups formed by oxidation ofpolysaccharide rings are not pendant aldehyde groups as defined herein.

Aldehyde-functionalized polysaccharides may be prepared by chemicallymodifying a polysaccharide to introduce pendant aldehyde groups. Usefulaldehyde-functionalized polysaccharides include, but are not limited to,aldehyde-functionalized derivatives of: dextran, carboxymethyldextran,starch, agar, cellulose, hydroxyethylcellulose, carboxymethylcellulose,pullulan, inulin, levan, and hyaluronic acid. The startingpolysaccharides are available commercially from sources such as SigmaChemical Co. (St. Louis, Mo.). Typically, polysaccharides are aheterogeneous mixture having a distribution of different molecularweights, and are characterized by an average molecular weight, forexample, the weight-average molecular weight (M_(w)) or the numberaverage molecular weight (M_(n)), as is known in the art. Therefore, thealdehyde-functionalized polysaccharides prepared from thesepolysaccharides are also a heterogeneous mixture having a distributionof different molecular weights. Suitable aldehyde-functionalizedpolysaccharides have a weight-average molecular weight of about 1,000 toabout 1,000,000 Daltons, more particularly about 3,000 to about 250,000Daltons, more particularly about 5,000 to about 60,000 Daltons, and moreparticularly about 7,000 to about 20,000 Daltons. In one embodiment, thealdehyde-functionalized polysaccharide is aldehyde-functionalizeddextran. In another embodiment, the aldehyde-functionalizedpolysaccharide is aldehyde-functionalized inulin.

Aldehyde-functionalized polysaccharides may be prepared using methodsknown in the art. Aldehyde-functionalized polysaccharides may beprepared using any of the methods described by Mehta et al. (WO99/07744). For example, dextran may be reacted with allyl glycidyl etherin an acid aqueous medium to form allyloxy dextran which is thenoxidized by ozonolysis to cleave the double bond and introduce aterminal aldehyde group, as described in detail in the Examples hereinbelow. Additionally, glycidol may be reacted with a polysaccharide, suchas dextran, in a basic aqueous medium to give an alkylatedpolysaccharide, as described by Chen (Biotechnology Techniques3:131-134, 1989). Periodate oxidation of the alkylated polysaccharideyields an aldehyde-functionalized polysaccharide having pendant aldehydegroups. The aldehyde-functionalized polysaccharides may also be preparedby the method described by Solarek et al. (U.S. Pat. No. 4,703,116)wherein a polysaccharide is reacted with a derivatizing acetal reagentin the presence of base and then the acetal is hydrolyzed by adjustingthe pH to less than 7.0.

Aldehyde-functionalized polysaccharides having dialdehyde functionalgroups can be prepared by first attaching a pendant group containingeither a terminal diene or by attaching a cyclic, disubstituted olefinto the polysaccharide ring. Attachment of the pendant groups can beaccomplished using a variety of methods, including reaction of thepolysaccharide with glycidyl ethers containing cyclic olefins orterminal dienes, or reaction with carboxylic acids or derivativesthereof which also contain cyclic olefins or terminal dienes. Oxidationof the polysaccharides derivatized with cyclic olefins or terminaldienes using methods known in the art, such as ozonolysis, yieldpolysaccharides derivatized with pendant dialdehydes.

The degree of aldehyde substitution may be determined using methodsknown in the art. For example, the degree of aldehyde substitution maybe determined by titrating the aldehyde-functionalized polysaccharidewith hydroxyl amine hydrochloride according to the method of Zhao andHeindel (Pharmaceutical Research 8:400, 1991). Suitablealdehyde-functionalized polysaccharides have a degree of aldehydesubstitution of about 10% to about 200%, more particularly about 30% toabout 200%, more particularly about 35% to about 120%, and moreparticularly about 40% to about 120%.

Water-Dispersible, Multi-Arm Amines:

Suitable water-dispersible, multi-arm amines include, but are notlimited to, water-dispersible multi-arm polyether amines,amino-terminated dendritic polyamidoamines, and multi-arm branched endamines. Typically, multi-arm amines suitable for use herein have anumber-average molecular weight of about 450 to about 200,000 Daltons,more particularly from about 2,000 to about 40,000 Daltons.

In one embodiment, the water-dispersible, multi-arm amine is a multi-armpolyether amine, which is a water-dispersible polyether having therepeat unit [—O—R]—, wherein R is a hydrocarbylene group having 2 to 5carbon atoms. Suitable multi-arm polyether amines include, but are notlimited to, amino-terminated star polyethylene oxides, amino-terminateddendritic polyethylene oxides, amino-terminated comb polyethyleneoxides, amino-terminated star polypropylene oxides, amino-terminateddendritic polypropylene oxides, amino-terminated comb polypropyleneoxides, amino-terminated star polyethylene oxide-polypropylene oxidecopolymers, amino-terminated dendritic polyethylene oxide-polypropyleneoxide copolymers, amino-terminated comb polyethylene oxide-polypropyleneoxide copolymers, and polyoxyalkylene triamines, sold under the tradename Jeffamine® triamines, by Huntsman LLC. (Houston, Tex.). Examples ofstar polyethylene oxide amines, include, but are not limited to, variousmulti-arm polyethylene glycol amines, and star polyethylene glycolshaving 3, 4, 6, or 8 arms terminated with primary amines (referred toherein as 3, 4, 6, or 8-arm star PEG amines, respectively). Examples ofsuitable Jeffamine® triamines include, but are not limited to,Jeffamine® T-403 (CAS No. 39423-51-3), Jeffamine® T-3000 (CAS No.64852-22-8), and Jeffamine® T-5000 (CAS No. 64852-22-8). In oneembodiment, the water-dispersible multi-arm polyether amine is aneight-arm polyethylene glycol having eight arms terminated by a primaryamine group and having a number-average molecular weight of about 10,000Daltons.

The multi-arm polyether amines are either available commercially, asnoted above, or may be prepared using methods known in the art. Forexample, multi-arm polyethylene glycols, wherein at least three of thearms are terminated by a primary amine group, may be prepared by puttingamine ends on multi-arm polyethylene glycols (e.g., 3, 4, 6, and 8-armstar polyethylene glycols, available from companies such as NektarTransforming Therapeutics; SunBio, Inc., Anyang City, South Korea; NOFCorp., Tokyo, Japan; or JenKem Technology USA, Allen, Tex.) using themethod described by Buckmann et al. (Makromol. Chem. 182:1379-1384,1981). In that method, the multi-arm polyethylene glycol is reacted withthionyl bromide to convert the hydroxyl groups to bromines, which arethen converted to amines by reaction with ammonia at 100° C. The methodis broadly applicable to the preparation of other multi-arm polyetheramines. Additionally, multi-arm polyether amines may be prepared frommulti-arm polyols using the method described by Chenault (copending andcommonly owned U.S. Patent Application Publication No.2007/0249870). Inthat method, the multi-arm polyether is reacted with thionyl chloride toconvert the hydroxyl groups to chlorine groups, which are then convertedto amines by reaction with aqueous or anhydrous ammonia. Other methodsthat may be used for preparing multi-arm polyether amines are describedby Merrill et al. in U.S. Pat. No. 5,830,986, and by Chang et al. in WO97/30103.

Water-dispersible, multi-arm amines suitable for use herein may also beamino-terminated dendritic polyamidoamines, sold under the trade nameStarburst® Dendrimers (available from Sigma-Aldrich, St Louis, Mo.).

In one embodiment, the water-dispersible, multi-arm amine is a multi-armbranched end amine, as described by Arthur (copending and commonly ownedInternational Patent Application Publication No. WO 2008/066787). Themulti-arm branched end amines are branched polymers having two or threeprimary amine groups at the end of each of the polymer arms. Themultiplicity of functional groups increases the statistical probabilityof reaction at a given chain end and allows more efficient incorporationof the branched molecules into a polymer network. The starting materialsused to prepare the branched end amines may be branched polymers such asmulti-arm polyether polyols including, but not limited to, comb and starpolyether polyols. The branched end amines can be prepared by attachingmultiple amine groups to the ends of the polymer by reaction with thehydroxyl groups using methods well known in the art. For example, abranched end amine having two amine functional groups on each end of thepolymer arms can be prepared by reacting the starting material, aslisted above, with thionyl chloride in a suitable solvent such astoluene to give the chloride derivative, which is subsequently reactedwith tris(2-aminoethyl)amine to give the branched end reactant havingtwo primary amine groups at the end of the polymer arms.

In one embodiment, the water-dispersible, multi-arm amine is aneight-arm branched end polyethylene glycol amine having two primaryamine groups at the end of the polymer arms and having a number-averagemolecular weight of about 10,000 Daltons.

In another embodiment, the water-dispersible, multi-arm amine is amixture of an eight-arm branched end polyethylene glycol amine havingtwo primary amine groups at the end of the polymer arms and having anumber-average molecular weight of about 10,000 Daltons, and aneight-arm polyethylene glycol amine having eight arms terminated by aprimary amine group and having a number-average molecular weight ofabout 10,000 Daltons.

It should be recognized that the water-dispersible, multi-arm amines aregenerally a somewhat heterogeneous mixture having a distribution of armlengths and in some cases, a distribution of species with differentnumbers of arms. When a multi-arm amine has a distribution of specieshaving different numbers of arms, it can be referred to based on theaverage number of arms in the distribution. For example, in oneembodiment the multi-arm amine is an 8-arm star PEG amine, whichcomprises a mixture of multi-arm star PEG amines, some having less thanand some having more than 8 arms; however, the multi-arm star PEG aminesin the mixture have an average of 8 arms. Therefore, the terms “8-arm”,“6-arm”, “4-arm” and “3-arm” as used herein to refer to multi-armamines, should be construed as referring to a heterogeneous mixturehaving a distribution of arm lengths and in some cases, a distributionof species with different numbers of arms, in which case the number ofarms recited refers to the average number of arms in the mixture.

Methods of Using the Hydrogel Tissue Adhesive

The hydrogel tissue adhesive disclosed herein may be used in variousforms. In one embodiment, the aldehyde-functionalized polysaccharidecontaining pendant aldehyde groups and the water-dispersible, multi-armamine are used as components of aqueous solutions or dispersions. Toprepare an aqueous solution or dispersion comprising analdehyde-functionalized polysaccharide (referred to herein as the “firstaqueous solution or dispersion”), at least one aldehyde-functionalizedpolysaccharide is added to water to give a concentration of about 5% toabout 40%, more particularly from about 5% to about 30%, and moreparticularly from about 10% to about 30% by weight relative to the totalweight of the solution or dispersion. Additionally, a mixture of atleast two different aldehyde-functionalized polysaccharides havingdifferent weight-average molecular weights, different degrees ofaldehyde substitution, or both different weight-average molecularweights and degrees of aldehyde substitution may be used. Where amixture of aldehyde-functionalized polysaccharides is used, the totalconcentration of the aldehyde-functionalized polysaccharides is about 5%to about 40% by weight, more particularly from about 5% to about 30%,and more particularly from about 10% to about 30% by weight relative tothe total weight of the solution or dispersion.

Similarly, to prepare an aqueous solution or dispersion comprising awater-dispersible, multi-arm amine (referred to herein as the “secondaqueous solution or dispersion”), at least one water-dispersible,multi-arm amine is added to water to give a concentration of about 5% toabout 70% by weight, more particularly from about 20% to about 50% byweight relative to the total weight of the solution or dispersion. Theoptimal concentration to be used depends on the intended application andon the concentration of the aldehyde-functionalized polysaccharide usedin the first aqueous solution or dispersion. Additionally, a mixture ofdifferent water-dispersible, multi-arm amines having differentnumber-average molecular weights, different numbers of arms, or bothdifferent number-average molecular weights and different numbers of armsmay be used. Where a mixture of water-dispersible, multi-arm amines isused, the total concentration of the multi-arm amines is about 5% toabout 70% by weight, more particularly from about 20% to about 50% byweight relative to the total weight of the solution or dispersion.

For use on living tissue, it is preferred that the first aqueoussolution or dispersion and the second aqueous solution or dispersion besterilized to prevent infection. Any suitable sterilization method knownin the art that does not adversely affect the ability of the componentsto react to form an effective hydrogel may be used, including, but notlimited to, electron beam irradiation, gamma irradiation, ethylene oxidesterilization, or filtration through a 0.2 μm pore membrane.

The first aqueous solution or dispersion and the second aqueous solutionor dispersion may further comprise various additives depending on theintended application. Preferably, the additive does not interfere witheffective gelation to form a hydrogel. The amount of the additive useddepends on the particular application and may be readily determined byone skilled in the art using routine experimentation. For example, thefirst aqueous solution or dispersion and/or the second aqueous solutionor dispersion may comprise at least one additive selected from pHmodifiers, antimicrobials, colorants, surfactants, pharmaceutical drugsand therapeutic agents.

The first aqueous solution or dispersion and/or the second aqueoussolution or dispersion may optionally include at least one pH modifierto adjust the pH of the solution(s) or dispersion(s). Suitable pHmodifiers are well known in the art. The pH modifier may be an acidic orbasic compound. Examples of acidic pH modifiers include, but are notlimited to, carboxylic acids, inorganic acids, and sulfonic acids.Examples of basic pH modifiers include, but are not limited to,hydroxides, alkoxides, nitrogen-containing compounds other than primaryand secondary amines, and basic carbonates and phosphates.

The first aqueous solution or dispersion and/or the second aqueoussolution or dispersion may optionally include at least one antimicrobialagent. Suitable antimicrobial preservatives are well known in the art.Examples of suitable antimicrobials include, but are not limited to,alkyl parabens, such as methylparaben, ethylparaben, propylparaben, andbutylparaben; triclosan; chlorhexidine; cresol; chlorocresol;hydroquinone; sodium benzoate; and potassium benzoate.

The first aqueous solution or dispersion and/or the second aqueoussolution or dispersion may optionally include at least one colorant toenhance the visibility of the solution(s) or dispersion(s). Suitablecolorants include dyes, pigments, and natural coloring agents. Examplesof suitable colorants include, but are not limited to, FD&C and D&Ccolorants, such as FD&C Violet No. 2, FD&C Blue No. 1, D&C Green No. 6,D&C Green No. 5, D&C Violet No. 2; and natural colorants such asbeetroot red, canthaxanthin, chlorophyll, eosin, saffron, and carmine.

The first aqueous solution or dispersion and/or the second aqueoussolution or dispersion may optionally include at least one surfactant.Surfactant, as used herein, refers to a compound that lowers the surfacetension of water. The surfactant may be an ionic surfactant, such assodium lauryl sulfate, or a neutral surfactant, such as polyoxyethyleneethers, polyoxyethylene esters, and polyoxyethylene sorbitan.

Additionally, the first aqueous solution or dispersion and/or the secondaqueous solution or dispersion may optionally include at least onepharmaceutical drug or therapeutic agent. Suitable drugs and therapeuticagents are well known in the art (for example see the United StatesPharmacopeia (USP), Physician's Desk Reference (Thomson Publishing), TheMerck Manual of Diagnosis and Therapy 18th ed., Mark H. Beers and RobertBerkow (eds.), Merck Publishing Group, 2006; or, in the case of animals,The Merck Veterinary Manual, 9th ed., Kahn, C. A. (ed.), MerckPublishing Group, 2005). Nonlimiting examples include anti-inflammatoryagents, for example, glucocorticoids such as prednisone, dexamethasone,budesonide; non-steroidal anti-inflammatory agents such as indomethacin,salicylic acid acetate, ibuprofen, sulindac, piroxicam, and naproxen;fibrinolytic agents such as a tissue plasminogen activator andstreptokinase; anti-coagulants such as heparin, hirudin, ancrod,dicumarol, sincumar, iloprost, L-arginine, dipyramidole and otherplatelet function inhibitors; antibodies; nucleic acids; peptides;hormones; growth factors; cytokines; chemokines; clotting factors;endogenous clotting inhibitors; antibacterial agents; antiviral agents;antifungal agents; anti-cancer agents; cell adhesion inhibitors; healingpromoters; vaccines; thrombogenic agents, such as thrombin, fibrinogen,homocysteine, and estramustine; radio-opaque compounds, such as bariumsulfate and gold particles and radiolabels.

Additionally, the second aqueous solution or dispersion comprising themulti-arm amine may optionally comprise at least one othermulti-functional amine having one or more primary amine groups toprovide other beneficial properties, such as hydrophobicity or modifiedcrosslink density. The multi-functional amine is capable of inducinggelation when mixed with an oxidized polysaccharide in an aqueoussolution or dispersion. The multi-functional amine may be a secondwater-dispersible, multi-arm amine, such as those described above, oranother type of multi-functional amine, including, but not limited to,linear and branched diamines, such as diaminoalkanes, polyaminoalkanes,and spermine; branched polyamines, such as polyethylenimine; cyclicdiamines, such as N, N′-bis(3-aminopropyl)piperazine,5-amino-1,3,3-trimethylcyclohexanemethylamine,1,3-bis(aminomethyl)cyclohexane, 1,4-diaminocyclohexane, andp-xylylenediamine; aminoalkyltrialkoxysilanes, such as3-aminopropyltrimethoxysilane and 3-aminopropyltriethoxysilane;aminoalkyldialkoxyalkylsilanes, such as3-aminopropyldiethoxymethylsilane, dihydrazides, such as adipicdihydrazide; linear polymeric diamines, such as linear polyethylenimine,α,ω-amino-terminated polyethers, α,ω-bis(3-aminopropyl)polybutanediol,β,ω-amino-terminated polyethers (linear Jeffamines®); comb polyamines,such as chitosan, polyallylamine, and polylysine, and di- andpolyhydrazides, such as bis(carboxyhydrazido)polyethers andpoly(carboxyhydrazido) star polyethers. Many of these compounds arecommercially available from companies such as Sigma-Aldrich and HuntsmanLLC. Typically, if present, the multi-functional amine is used at aconcentration of about 5% by weight to about 1000% by weight relative tothe weight of the multi-arm amine in the aqueous solution or dispersion.

When the first aqueous solution or dispersion and the second aqueoussolution or dispersion are mixed they react to form a crosslinkedhydrogel composition comprising at least one aldehyde-functionalizedpolysaccharide containing pendant aldehyde groups; and at least onewater-dispersible, multi-arm amine wherein at least three of the armsare terminated by at least one primary amine group, and wherein the atleast one aldehyde-functionalized polysaccharide and the at least onewater-dispersible, multi-arm amine are crosslinked through covalentbonds formed between the pendant aldehyde groups of thealdehyde-functionalized polysaccharide and the primary amine groups ofthe water-dispersible, multi-arm amine. The covalent bonds may be imine,aminal or hemiaminal bonds. The degradation time of the hydrogel may betuned for the needs of the intended application by using differentamounts of the aldehyde-functionalized polysaccharide in the firstaqueous solution or dispersion and the water-dispersible, multi-armamine in the second aqueous solution or dispersion in terms of weightpercent and/or by altering the amount of funtionalization of eitheramine on the water-dispersible, multi-arm amine or aldehyde on thealdehyde-functionalized polysaccharide, as shown in the Examples hereinbelow.

The first aqueous solution or dispersion and the second aqueous solutionor dispersion may be used to apply a coating to an anatomical site ontissue of a living organism. The two aqueous solutions or dispersionsmay be applied to the site in any number of ways. Once both solutions ordispersions are combined on a site, they crosslink to form a hydrogelwhich provides a coating on the site.

In one embodiment, the two aqueous solutions or dispersions are appliedto the site sequentially using any suitable means including, but notlimited to, spraying, brushing with a cotton swab or brush, or extrusionusing a pipette, or a syringe. The solutions or dispersions may beapplied in any order. Then, the solutions or dispersions are mixed onthe site using any suitable device, such as a cotton swab, a spatula, orthe tip of the pipette or syringe.

In another embodiment, the two aqueous solutions or dispersions aremixed manually before application to the site. The resulting mixture isthen applied to the site before it completely cures using a suitableapplicator, as described above.

In another embodiment, the first aqueous solution or dispersion and thesecond aqueous solution or dispersion are applied to the sitesimultaneously where they mix to form a hydrogel. For example, the twoaqueous solutions or dispersions may be contained in separate barrels ofa double-barrel syringe. In this way the two aqueous solutions ordispersions are applied simultaneously to the site with the syringe.Suitable double-barrel syringe applicators are known in the art. Forexample, Redl describes several suitable applicators for use in theinvention in U.S. Pat. No. 6,620,125, (particularly FIGS. 1, 5, and 6,which are described in Columns 4, line 10 through column 6, line 47).The two aqueous solutions or dispersions may also be applied to the siteusing a dual-lumen catheter, such as those available from Bistech, Inc.(Woburn, Mass.). Additionally, injection devices for introducing twoliquid components endoscopically into the body simultaneously are knownin the art and may be adapted for the delivery of the two aqueoussolutions or dispersions disclosed herein (see for example, Linder etal., U.S. Pat. No. 5,322,510).

In another embodiment, the first aqueous solution or dispersion and thesecond aqueous solution or dispersion may be premixed and delivered tothe site using a double barrel syringe containing a motionless mixer,such as that available from ConProtec, Inc. (Salem, N.H.) or MixpacSystems AG (Rotkreuz, Switzerland). Alternatively, the mixing tip may beequipped with a spray head, such as that described by Cruise et al. inU.S. Pat. No. 6,458,147. Additionally, the mixture of the two aqueoussolutions or dispersions from the double-barrel syringe may be appliedto the site using a catheter or endoscope. Devices for mixing a twoliquid component tissue adhesive and delivering the resulting mixtureendoscopically are known in the art and may be adapted for the mixingand delivery of the two aqueous solutions or dispersions disclosedherein (see for example, Nielson, U.S. Pat. No. 6,723,067; and Redl etal., U.S. Pat. No. 4,631,055).

In another embodiment, the two aqueous solutions or dispersions may beapplied to the site using a spray device, such as those described byFukunaga et al. (U.S. Pat. No. 5,582,596), Delmotte et al. (U.S. Pat.No. 5,989,215) or Sawhney (U.S. Pat. No. 6,179,862).

In another embodiment, the two aqueous solutions or dispersions may beapplied to the site using a minimally invasive surgical applicator, suchas those described by Sawhney (U.S. Pat. No. 7,347,850).

In another embodiment, the hydrogel tissue adhesive of the invention isused to bond at least two anatomical sites together. In this embodiment,the first aqueous solution or dispersion is applied to at least oneanatomical site, and the second aqueous solution or dispersion isapplied to at least one of either the same site or one other site usingthe methods described above. The two or more sites are contacted andheld together manually or using some other means, such as a surgicalclamp, for a time sufficient for the mixture to cure. Alternatively, amixture of the two aqueous solutions or dispersions is applied to atleast one of the anatomical sites to be bonded using methods describedabove. The two or more sites are contacted and held together manually orusing some other means, such as a surgical clamp, for a time sufficientfor the mixture to cure.

In another embodiment, the aldehyde-functionalized polysaccharide, andthe water-dispersible, multi-arm amine may be used in the form of finelydivided powders. The powders may be prepared using any suitable method.For example, each of the aqueous solutions or dispersions describedabove may be dried using heat, vacuum, a combination of heat and vacuum,or by lyophilization, to form powders. Optionally, the powders may becomminuted into finer particles using methods known in the artincluding, but not limited to, grinding, milling, or crushing with amortar and pestle. The finely divided powders may be sterilized usingthe methods described above. The finely divided powders may be appliedto an anatomical site on tissue of a living organism in a variety ofways. For example, the powders may be individually applied to the sitein any order by sprinkling or spraying. Additionally, the powders may bepremixed and the resulting mixture applied to the site by sprinkling orspraying. The powders may be hydrated on the site by the addition of anaqueous solution such as water or a suitable buffer (e.g.,phosphate-buffered saline) or by the physiological fluids present at thesite. The finely divided powders may also be used to bond two anatomicalsites together as described above for the aqueous solutions ordispersions. Alternatively, the powders may be hydrated with water or asuitable aqueous solution prior to use to form the first and secondaqueous solutions or dispersions, described above.

In another embodiment, the hydrogel tissue adhesive disclosed herein maybe used in the form of a dried hydrogel. In this embodiment, a driedhydrogel is prepared by combining in a solvent at least onealdehyde-functionalized polysaccharide with at least onewater-dispersible, multi-arm amine to form a hydrogel, and treating thehydrogel to remove at least a portion of the solvent to form the driedhydrogel. Suitable solvents include, but are not limited to, water,ethanol, isopropanol, tetrahydrofuran, hexanes, polyethylene glycol, andmixtures thereof. If two different solvents are used, the two solventsare miscible with each other. In one embodiment the solvent is water.The aldehyde-functionalized polysaccharide and the water-dispersible,multi-arm amine may be combined in various ways. For example, the firstaqueous solution or dispersion comprising the aldehyde-functionalizedpolysaccharide and the second aqueous solution or dispersion comprisingthe water-dispersible, multi-arm amine, may be prepared and mixed asdescribed above to form the hydrogel. The solutions or dispersions usedto prepare the hydrogel may further comprise various additives dependingon the intended application. Any of the additives described above may beused. The hydrogel is then treated to remove at least a portion of thesolvent contained therein to form the dried hydrogel. Preferably,substantially all of the solvent is removed from the hydrogel. Thesolvent may be removed from the hydrogel using methods known in the art,for example, using heat, vacuum, a combination of heat and vacuum, orflowing a stream of dry air or a dry inert gas such as nitrogen over thehydrogel. The dried hydrogel may be sterilized using the methodsdescribed above. The dried hydrogel may be applied to an anatomical sitein a number of ways, as described below. The dried hydrogel may behydrated on the site by the addition of a suitable aqueous solution suchas water or a buffer (e.g., phosphate-buffered saline) or by thephysiological fluids present at the site.

In one embodiment, the dried hydrogel may be used in the form of a film.The dried hydrogel film may be formed by casting a mixture of thesolutions or dispersions, as described above, on a suitable substrateand treating the resulting hydrogel to form a dried hydrogel film. Thedried hydrogel film may be applied directly to an anatomical site.Additionally, the dried hydrogel film may be used to bond two anatomicalsites together.

In another embodiment, the dried hydrogel may be used in the form offinely divided particles. The dried hydrogel particles may be formed bycomminuting the dried hydrogel using methods known in the art,including, but not limited to, grinding, milling, or crushing with amortar and pestle. The dried hydrogel particles may be applied to ananatomical site in a variety of ways, such as sprinkling or spraying,and may also be used to bond two anatomical sites together.

Kits

In one embodiment, the invention provides a kit comprising at least onealdehyde-functionalized polysaccharide containing pendant aldehydegroups and at least one water-dispersible, multi-arm amine wherein atleast three of the arms are terminated by at least one primary aminegroup.

In another embodiment, the kit comprises a first aqueous solution ordispersion comprising at least one aldehyde-functionalizedpolysaccharide containing pendant aldehyde groups and a second aqueoussolution or dispersion comprising at least one water-dispersible,multi-arm amine wherein at least three of the arms are terminated by atleast one primary amine group. Each of the aqueous solutions ordispersions may be contained in any suitable vessel, such as a vial or asyringe barrel.

In another embodiment, the kit comprises at least onealdehyde-functionalized polysaccharide containing pendant aldehydegroups and at least one water-dispersible, multi-arm amine wherein atleast three of the arms are terminated by at least one primary aminegroup in the form of finely divided powders, as described above. Thepowders may be contained in separate containers or they may be premixedand contained in a single container. The kit may also comprise anaqueous solution for hydrating the powders.

In another embodiment, the kit comprises a dried hydrogel as describedabove. The dried hydrogel may be in the form of a film, finely dividedparticles, or other dried forms. The kit may further comprise an aqueoussolution for hydrating the dried hydrogel. The dried hydrogel particlesmay be contained in any suitable container.

Medical Applications:

The hydrogel disclosed herein may be useful as a tissue adhesive orsealant for medical applications that require a more rapid degradationtime, including but not limited to, prevention of undesired tissue-totissue adhesions resulting from trauma or surgery. In theseapplications, the aldehyde-functionalized polysaccharide and thewater-dispersible, multi-arm amine, or the dried hydrogel may be appliedto the desired anatomical site using the methods described above.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

Reagent Preparation Preparation of Dextran Having Pendant AldehydeGroups (AFD-15-90):

Dextran containing pendant aldehyde groups and having a weight-averagemolecular weight of about 15 kDa and a degree of aldehyde substitutionof about 90% was prepared using a two step procedure. In the first step,dextran having a weight-average molecular weight of about 9 to about 11kDa was reacted with allyl glycidyl ether to form an olefinintermediate, which was then reacted with ozone to form the dextrancontaining pendant aldehyde groups.

In the first step, 30 g of dextran (average molecular weight of 9-11kDa, Sigma), and 30 mL of water were added into a 3-neck flask. Thesolution was cooled to 10° C. and then 39 mL of a 20 wt % NaOH solutionwas added. The resulting solution was stirred for 25 min, giving a faintyellow solution, and then 84.58 g (4 equiv) of allyl glycidyl ether(Aldrich) was added. The resulting mixture was heated to 65° C. for 5.5hours, and then allowed to cool to room temperature, after which the pHwas adjusted to 7.0 with 1 N HCl. The mixture was diluted with anadditional 200 mL of water and purified using a Millipore Pellicon IIultrafiltration system (Millipore Corp., Billerica, Mass.), byfiltration through 1 kDa cutoff filters with continuous replacement offiltrate with pure water until 5× the initial solution volume had beencollected as filtrate. A small sample was taken and lyophilized foranalytical purposes. The remainder of the filtrate was used directly inthe subsequent ozonolysis step. The degree of substitution wasdetermined to be 1.33 by proton NMR from the ratio of integrationbetween the olefin peaks and the anomeric peaks at 4.8-5.0.

In the second step, 460 mL of the filtrate resulting from step 1 wasadded to a 3-neck, 2 L flask equipped with magnetic stir bar, and spargetube inlet. The solution was cooled in an ice bath to 0-5° C. and thensparging with ozone was begun from an ozone generator (ClearWater Tech,LLC., San Luis Obispo, Calif.; Model CD10) at 100% power which generates7% ozone. Foaming occurred, which was controlled by the addition of afew drops of 1-heptanol. Samples were taken at 6.25 and 7.75 hours andanalyzed using 13-C NMR. Disappearance of resonances at 118 and 134 ppmindicated that the olefin had been consumed after 7.75 hours. Then, asolution of sodium sulfite (23.3 g in 138 mL water) was added dropwisewith stirring. A slight exotherm was observed and the reaction mixturewas allowed to stir overnight under nitrogen. The mixture wastransferred to a 1 L glass jar and filtered through a Millipore PelliconII ultrafiltration system with 1 kDa cutoff filters. Water wascontinuously added to replace the filtrate collected, and the retentatewas recycled back to the glass jar. This process was continued untilmore than 5× the initial reaction volume had been collected in thefiltrate. The purified solution was then frozen and lyophilized to give53.9 g of a fluffy white solid. The degree of aldehyde substitution ofthe resulting solid product was determined to be 89% using the method ofZhao and Heindel (Pharmaceutical Research 8:400, 1991). Theweight-average molecular weight of the aldehyde-functionalized dextranwas determined to be about 15 kDa using size exclusion chromatography(SEC). This aldehyde-functionalized dextran is referred to herein asAFD-15-90.

A second preparation of this aldehyde-functionalized dextran was madeusing the same procedure. The degree of aldehyde substitution of theresulting product was determined to be 92% using the method of Zhao andHeindel and the weight-average molecular weight was found to be 16 kDausing SEC. This aldehyde-functionalized dextran is referred to herein asAFD-16-92.

Preparation of Inulin Having Pendant Aldehyde Groups (AFI-12-49):

Inulin containing pendant aldehyde groups and having a weight-averagemolecular weight of about 12 kDa and a degree of aldehyde substitutionof about 49% was prepared using the two step procedure described above.

In the first step, 20 g of inulin (average molecular weight of about 4kDa, Sigma) was suspended in 200 mL of water, heated to 70° C. for 1hour to dissolve, and then cooled to 65° C. To this solution was added23 mL of sodium hydroxide solution (20 wt % in water), followed by theslow addition of allyl glycidyl ether (50.7 g) via a syringe pump at arate of 3 mL/min. After the addition, the mixture was heated to 65° C.for 6 hours. After which time, the reaction mixture was cooled to roomtemp and neutralized to pH 7 with 50% HCl. The reaction mixture waspurified by ultrafiltration over a 1000 MWCO membrane (collect 10×volume of waste).

¹H NMR (D₂O): d 5.95 ppm (m, integral 1.0, OCH₂C(H)═CH₂), 5.31 (dd,integral 2.1, OCH2(CH)═CH₂), 4.37 (br. s, integral 0.40), 4.25 (br. s,integral 0.76), 4.0-3.55 (br. m, integral 12.9).

In the second step, the filtrate was cooled to approximately 5° C. usingan ice/water bath. Ozone was sparged into the stirred solution for 6hours. 1-Heptanol was added (a few drops) to control foaming. At the endof the reaction, sodium sulfite solution (16 g in 100 mL water) wasadded to the cooled solution (cooled in an ice/water bath). The solutionwas stirred at room temperature overnight. The product was purified byultrafiltration (MWCO 1000, collect 12× volume of waste). The filtratewas lyophilized to yield a white solid.

Size exclusion chromatography (SEC) analysis of the product gave thefollowing: M_(w)=1.2×10⁴, M_(n)=7.3×10³, M_(z)=1.5×10⁴, M_(w)/M_(n)=1.6.

The degree of aldehyde substitution was determined to be about 49% bytitration of the hydroxylamine adduct of the inulin aldehyde using themethod described by Zhao and Heindel (Pharmaceutical Research 8:400,1991). This aldehyde-functionalized inulin is referred to herein asAFI-12-49.

Preparation of Dextran Having Pendant Aldehyde Groups (AFD-7-86):

Dextran containing pendant aldehyde groups and having a weight-averagemolecular weight of about 5 to 11 kDa and a degree of aldehydesubstitution of 86% was prepared using a two step procedure. In thefirst step, dextran having a weight-average molecular weight of about 5to about 11 kDa was reacted with glycidol to form alkylated dextran. Inthe second step the alkylated dextran was oxidized with sodium periodateto oxidize the terminal diol groups added in the first step to givedextran having pendant aldehyde groups.

In the first step, 20 g of dextran (average molecular weight of 5-11kDa, Sigma), was suspended in 20 mL of water and heated to 55° C. Tothis solution was added 25 mL of sodium hydroxide solution (20 wt % inwater), followed by the slow addition of glycidol (36 g, Aldrich) usinga syringe pump at a flow rate of 1.0 mL/min at 55° C. Then, the mixturewas heated to 55° C. for 6 hours, after which the reaction mixture wascooled to room temperature. The product was washed twice with 20 mL ofether to remove excess reagent. The resulting yellow homogeneous mixturewas neutralized with 50% HCl over ice (final pH was 7.3). The sample wasprecipitated in approximately 5× volume of cold isopropanol (˜0° C.).The isopropanol layer was decanted off, the solid product washed withcold isopropanol, and the process of dissolution followed byprecipitation was repeated two more times. The solid product was driedunder vacuum for 48 hours.

In the second step, 15 g of the solid product from the first step wasdissolved in 150 mL of water in a round bottom flask and then theresulting solution was cooled to 4° C. Sodium periodate solution (8.25 gin 85 mL of water) was added to the round bottom flask dropwise over 30min. The reaction mixture was stirred at 4° C. for 2 hours, and then10.4 g (9.3 mL) of ethylene glycol was added to the reaction mixture,which was then is stirred for 10 min. The product was purified using aTFF System (Millipore Corp., Billerica, Mass.) with a 1000 molecularweight cut off membrane and freeze dried to yield 10 g of white powder.The degree of aldehyde substitution of the product was determined to be86% by titration of the hydroxylamine adduct using the method describedby Zhao and Heindel (Pharmaceutical Research 8:400, 1991).

Size exclusion chromatography (SEC) analysis of the product gave thefollowing: M_(w)=7.4×10³, M_(n)=4.8×10³, M_(z)=1.1×10⁴, M_(w)/M_(n)=1.5.This aldehyde-functionalized dextran is referred to herein as AFD-7-86.

Preparation of Dextran Having Pendant Aldehyde Groups (AFD-9-120):

Dextran containing pendant aldehyde groups and having a weight-averagemolecular weight of about 9 kDa and a degree of aldehyde substitution of120% was prepared using a two step procedure. In the first step, dextranhaving a weight-average molecular weight of about 5 to about 11 kDa wasreacted with glycidol to form alkylated dextran. In the second step thealkylated dextran was oxidized with sodium periodate to oxidize theterminal diol groups added in the first step to give dextran havingpendant aldehyde groups.

In the first step, three batches of dextran were reacted with glycidolto form the alkylated dextran. In two of the batches, 20 g of dextran(average molecular weight of 5-11 kDa, Sigma), was reacted with glycidol(36 g, Aldrich) in the same manner as described above, for the firststep of the preparation of AFD-7-86. In the third batch, the mole ratioof glycidol to dextran was varied. Dextran (10 g) was dissolved in asolution formed by combining 10 mL of water and 12.5 mL sodium hydroxidesolution (20 wt %). Glycidol (44 g, Aldrich) was added to the dextransolution using a syringe pump at a rate of 0.7 mL/mn. The mixture washeated to 55° C. and 20 mL of water was added. The reaction mixture washeated at 55° C. for 6 hours. The product was isolated as describedabove for AFD-7-86. The three batches were combined to make a masterbatch of materials for the second step.

In the second step, 25 g of the solid product from the master batchdescribed above was dissolved in 250 mL of water in a round bottom flaskand then the resulting solution was cooled to 4° C. Sodium periodatesolution (20.8 g in 125 mL of water) was added to the round bottom flaskdropwise over 1 hour. The reaction mixture was stirred at 4° C. for 2hours, and then 66 g of ethylene glycol was added to the reactionmixture, which was then stirred for 30 min. After which time, thereaction mixture was filtered. The filtrate was purified using a TFFSystem (Millipore Corp., Billerica, Mass.) with a 1000 molecular weightcut off membrane and freeze dried to yield 17 g of white powder. Thedegree of aldehyde substitution of the product was determined to be 120%by titration of the hydroxylamine adduct using the method described byZhao and Heindel (Pharmaceutical Research 8:400, 1991).

Size exclusion chromatography (SEC) analysis of the product gave thefollowing: M_(w)=8.9×10³, M_(n)=6.8×10³, M_(z)=1.2×10⁴, M_(w)/M_(n)=1.3.This aldehyde-functionalized dextran is referred to herein as AFD-9-120.

Preparation of Dextran Having Pendant Aldehyde Groups (AFD-13-64):

Dextran containing pendant aldehyde groups and having a weight-averagemolecular weight of about 13 kDa and a degree of aldehyde substitutionof 64% was prepared using a two step procedure. In the first step,dextran having a weight-average molecular weight of about 5 to about 11kDa was reacted with glycidol to form alkylated dextran. In the secondstep the alkylated dextran was oxidized with sodium periodate to oxidizethe terminal diol groups added in the first step to give dextran havingpendant aldehyde groups.

The first step was carried out as described above for AFD-9-120. In thesecond step, 25 g of the solid product from the master batch of thealkylated dextran described above for AFD-9-120 was dissolved in 250 mLof water in a round bottom flask and then the resulting solution wascooled to 4° C. Sodium periodate solution (10.4 g in 60 mL of water) wasadded to the round bottom flask dropwise over 1 hour. The reactionmixture was stirred at 4° C. for 2 hours, and then 6 g of ethyleneglycol was added to the reaction mixture, which was then stirred for 30min. Then the reaction mixture was filtered. The filtrate was purifiedusing a TFF System (Millipore Corp., Billerica, Mass.) with a 1000molecular weight cut off membrane and freeze dried to yield 17 g ofwhite powder. The degree of aldehyde substitution of the product wasdetermined to be 64% by titration of the hydroxylamine adduct using themethod described by Zhao and Heindel (Pharmaceutical Research 8:400,1991).

Size exclusion chromatography (SEC) analysis of the product gave thefollowing: M_(w)=1.3×10⁴, M_(n)=9.8×10³, M_(z)=1.8×10⁴, M_(w)/M_(n)=1.3.This aldehyde-functionalized dextran is referred to herein as AFD-13-64.

Preparation of Dextran Having Pendant Aldehyde Groups (AFD-13-46):

Dextran containing pendant aldehyde groups and having a weight-averagemolecular weight of about 13 kDa and a degree of aldehyde substitutionof 46% was prepared using a two step procedure. In the first step,dextran having a weight-average molecular weight of about 9 to about 11kDa was reacted with allyl glycidyl ether to form an olefinintermediate, which was then reacted with ozone to form the dextrancontaining pendant aldehyde groups.

In the first step, 30 g of dextran (average molecular weight of 9-11kDa, Sigma), and 30 mL of water were added into a 3-neck flask. Thesolution was cooled to 10° C. and then 39 mL of a 20 wt % NaOH solutionwas added. The resulting solution was stirred for 25 min, giving a faintyellow solution, and then 42.27 g (2 equiv) of allyl glycidyl ether(Aldrich) was added. The resulting mixture was heated to 65° C. for 5.5hours and then allowed to cool to room temperature, after which the pHwas adjusted to 7.0 with 1 N HCl. The mixture was diluted with anadditional 200 mL of water and purified with a Millipore Pellicon IIultrafiltration system (Millipore Corp., Billerica, Mass.) using 1 kDacutoff filters with continuous replacement of filtrate with pure wateruntil 5× the initial solution volume had been collected as filtrate. Asmall sample of the filtrate was taken and lyophilized for analyticalpurposes. The remainder of the filtrate was used directly in thesubsequent ozonolysis step. The degree of substitution was found to be0.84 by NMR, integrating the olefinic peaks relative to the anomericresonances.

In the second step, 475 mL of the filtrate resulting from step 1 wasadded to a 3-neck, 2 L flask equipped with magnetic stir bar, and spargetube inlet. The solution was cooled in an ice bath to 0-5° C. and thensparging with ozone was begun from an ozone generator (ClearWater Tech,LLC., San Luis Obispo, Calif.; Model CD10) at 100% power which generates7% ozone. Foaming occurred, which was controlled by the addition of afew drops of 1-heptanol. Samples were taken at 2 and 4 hours andanalyzed using 13-C NMR. Disappearance of resonances at 118 and 134 ppmindicated that the olefin had been consumed after 4 hours. Then, asolution of sodium sulfite (19.6 g in 117 mL of water) was addeddropwise with stirring. A slight exotherm was observed and the reactionmixture was allowed to stir overnight under nitrogen. The mixture wastransferred to a 1 L glass jar and filtered through a Millipore PelliconII ultrafiltration system with 1 kDa cutoff filters. Water wascontinuously added to replace the filtrate collected, and the retentatewas recycled back to the glass jar. This procedure was continued untilmore than 5× the initial reaction volume had been collected in thefiltrate. The purified solution was then frozen and lyophilized to give28.6 g of a fluffy white solid. The degree of aldehyde substitution ofthe resulting solid product was determined to be 46% using the method ofZhao and Heindel (Pharmaceutical Research, 1991, 8:400). Theweight-average molecular weight of the aldehyde-functionalized dextranwas determined to be about 13 kDa using size exclusion chromatography(SEC). This aldehyde-functionalized dextran is referred to herein asAFD-13-46.

Preparation of Dextran Having Pendant Aldehyde Groups (AFD-19-64):

Dextran containing pendant aldehyde groups and having a weight-averagemolecular weight of about 19 kDa and a degree of aldehyde substitutionof 60% was prepared using the two step procedure described above.

In the first step, 30 g of dextran (average molecular weight of 9-11kDa, Sigma), and 30 mL of water were added into a 3-neck flask. Thesolution was cooled to 10° C. and then 39 mL of a 20 wt % NaOH solutionwas added. The resulting solution was stirred for 25 min, giving a faintyellow solution, and then 63.41 g (3 equiv) of allyl glycidyl ether(Aldrich) was added. The resulting mixture was heated to 65° C. for 5.5hours, and then allowed to cool to room temperature, after which the pHwas adjusted to 7.0 with 1 N HCl. The mixture was diluted with anadditional 200 mL of water and purified using the Millipore Pellicon IIultrafiltration system, by filtration through 1 kDa cutoff filters withcontinuous replacement of filtrate with pure water until 5× the initialsolution volume had been collected as filtrate. A small sample was takenand lyophilized for analytical purposes. The remainder of the filtratewas used directly in the subsequent ozonolysis step. The degree ofsubstitution was determined to be 0.99 by NMR from the ratio ofintegration between the olefin peaks and the anomeric peaks at 4.8-5.0.

In the second step, 475 mL of the filtrate resulting from step 1 wasadded to a 3-neck, 2 L flask equipped with magnetic stir bar, and spargetube inlet. The solution was cooled in an ice bath to 0-5° C. and thensparging with ozone was begun from an ozone generator (ClearWater Tech,LLC., San Luis Obispo, Calif.; Model CD10) at 100% power which generates7% ozone. Foaming occurred, which was controlled by the addition of afew drops of 1-heptanol. Samples were taken at 4 and 5.75 hours andanalyzed using 13-C NMR. Disappearance of resonances at 118 and 134 ppmindicated that the olefin had been consumed after 5.75 hours. Then, asolution of sodium sulfite (23.3 g in 139 mL water) was added dropwisewith stirring. A slight exotherm was observed and the reaction mixturewas allowed to stir overnight under nitrogen. The mixture wastransferred to a 1 L glass jar and filtered through a Millipore PelliconII ultrafiltration system with 1 kDa cutoff filters. Water wascontinuously added to replace the filtrate collected, and the retentatewas recycled back to the glass jar. This process was continued untilmore than 5× the initial reaction volume had been collected in thefiltrate. The purified solution was then frozen and lyophilized to give39.1 g of a fluffy white solid. The degree of aldehyde substitution ofthe resulting solid product was determined to be 60% using the method ofZhao and Heindel (Pharmaceutical Research, 1991, 8:400). Theweight-average molecular weight of the aldehyde-functionalized dextranwas determined to be about 19 kDa using size exclusion chromatography(SEC). This aldehyde-functionalized dextran is referred to herein asAFD-19-64.

Preparation of Dextran Having Pendant Dialdehyde Groups (DAFD-10-16):

Dialdehyde-functionalized dextran in which the pendant dialdehyde groupsare linked to the dextran backbone by an ether bond was prepared using athree step procedure.

Step 1:

To a 300 mL, two-neck flask equipped with a magnetic stir bar andnitrogen inlet was added 74.3 mL of a 40 wt % sodium hydroxide solutionand 0.94 g (2.91 mmol) of tetrabutylammonium bromide. The solution wascooled to 5-10° C. and treated with 5.0 g (59.44 mmol) of3-cyclopentene-1-ol followed by dropwise addition of epichlorohydrin22.0 g (237.76 mmol) over a 20 min period. The reaction mixture wasstirred at room temperature overnight. Then, the reaction mixture waspoured onto approximately 50 mL of ice/water and stirred to give asolution, which was extracted three times with 50 mL portions of diethylether. The combined organic layers were washed with brine solution untilneutral to litmus and dried over MgSO₄. The solvent was removed on arotary evaporator to yield a brown liquid. The crude product waspurified by distillation. The product was collected at 118° C. and 25 mmHg (3.3 kPa).

¹H NMR in CDCl₃ δ ppm (2.42, m, 2H; 2.5, m 1H; 2.6, m, 2H, 2.78, m, 1H;3.15, m, 1H; 3.4, m, 1H; 3.65-3.68, m, 1H; 4.26, m, 1H; 5.68 s, 2H) Step2:

Into a 10 mL, 2-neck flask equipped with a magnetic stir bar and refluxcondenser with nitrogen inlet was placed 0.58 mL of water followed by0.5778 g (3.567 mmol) of dextran having a weight-average molecularweight of about 9 to about 11 kDa. The solution was stirred to form asuspension. To the flask was added 0.75 mL of 20 wt % NaOH solution.After stirring for 30 min, 1.0 g (7.134 mmol) of glycidyl3-cyclopentenyl ether was added. The solution was heated in an oil bathat 65° C. for 5.5 hours. Then, the reaction mixture was cooled to roomtemperature and the pH was adjusted to 7.0 with 0.5 M HCl. The resultingsolution was diluted to approximately 400 mL with water, then purifiedusing the Millipore Pellicon II ultrafiltration system with a 1000molecular weight cutoff cassette. A small aliquot was lyophilized foranalysis.

¹H NMR was obtained in D₂O. δ ppm (2.2, d; 2.4, d; 3.31-3.7,m; 4.1, s;4.77, s; 4.9, s(b); 5.55, s)

The remainder of the solution was treated with ozone to makefunctionalized dextran. From NMR, the degree of substitution wasdetermined by integration of the anomeric peaks at 5.0-5.2 ppm vs. theolefinic peaks. The degree of substitution was found to be 0.52.

Step 3:

A 1 L, 3-neck flask containing 400 mL of the aqueous solution offunctionalized dextran (from the previous step) at approximately 8° C.was sparged with ozone for 5.5 hours. The ozone flow was stopped and asolution of 0.45 g of sodium sulfite in 3 mL of water was added to theflask at 8° C. The solution was stirred overnight and purified byultrafiltration on the Millipore Pellicon II ultrafiltration systemusing 1000 MWCO cassette. The final solution was frozen and lyophilizedto yield 0.68 g of a foam-like solid.

¹H NMR was obtained in D₂O. δ ppm (1.318, m; 1.523, m; 1.8, m; 2.1, m;2.3-2.48, m; 3.3-3.9, m; 4.9, s; 5.1, s(b); 5.5, s)

The degree of aldehyde substitution was determined to be about 16% bytitration of the hydroxylamine adduct of the dialdehyde-functionalizeddextran using the method described by Zhao and Heindel (PharmaceuticalResearch 8:400, 1991). This dialdehyde-functionalized dextran isreferred to herein as DAFD-10-16.

Preparation of Dextran Having Pendant Dialdehyde Groups (DAFD-EsterLinked):

Dialdehyde-functionalized dextran in which the pendant dialdehyde groupsare linked to the dextran backbone by an ester bond was prepared using atwo step procedure.

Step 1:

Into a 50 mL, 3-neck flask equipped with magnetic stir bar and a refluxcondenser was placed 22 mL of dimethylacetamide (DMAC) followed by 1.817g (11.217 mmol) of dextran having a weight-average molecular weight ofabout 9 to about 11 kDa and 1.09 g (25.71 mmol) of lithium chloride. Asuspension formed, which was heated to 90° C. for one hour until a clearsolution resulted. The solution was cooled to room temperature and 0.91mL (11.217 mmol) of pyridine was added followed by dropwise addition of2.18 g (11.217 mmol) of 3-cyclopentene carbonyl chloride.4-Dimethylaminopyridine (DMAP, Aldrich) (30 mg) was added and themixture was heated mixture at 60° C. overnight i.e., approximately 20hours). After cooling, the resulting brown solution was added dropwiseto 200 mL of cooled water with stirring to give a yellow solution. ThepH was adjusted from 2.15 to 6.0 using 0.25 N NaOH solution. The crudeproduct was purified on a Millipore Pellicon II ultrafiltration systemusing a 1000 molecular weight cutoff cassette. A small aliquot wasfrozen and lyophilized to obtain an analytical sample.

¹H NMR. δ ppm (2.59-2.66, m); (2.85, s); (3.0, s); (3.24(b), s);(3.46-3.51, m); (3.52-3.71, m); (3.84, d); (3.93, d); (4.91, s); (4.97,s); (5.12, t); (5.68, s) Step 2:

A 1 L, 3-neck flask equipped with a magnetic stir bar and containing 350mL of the solution resulting from the previous step at approximately 8°C. was sparged with an ozone stream for 5 hours. After discontinuing theozone sparge, a solution of 1.41 g of sodium sulfite in 8.4 mL water wasadded. The resulting mixture was stirred at room temperature overnight,then purified on a Millipore Pellicon II ultrafiltration system using a1000 molecular weight cutoff cassette filter. The solution was frozenand lyophilized to yield 1.92 g of a white solid.

¹H NMR was submitted in D₂O. δ ppm (3.49-3.79, m); (3.89-3.97, m); (4.97d, (broad)); 5.16 (s, (b))

This dialdehyde-functionalized dextran is referred to herein asDAFD-Ester Linked.

Preparation of Oxidized Dextran (D10-50)

Dextran aldehyde is made by oxidizing dextran in aqueous solution withsodium metaperiodate. An oxidized dextran having an average molecularweight of about 10,000 Da and an oxidation conversion of about 50%(i.e., about half of the glucose rings in the dextran polymer areoxidized to dialdehydes) is prepared from dextran having aweight-average molecular weight of 8,500 to 11,500 Daltons (Sigma) bythe method described by Cohen et al. (copending and commonly ownedInternational Patent Application Publication No. WO 2008/133847). Atypical procedure is described here.

A 20-L reactor equipped with a mechanical stirrer, addition funnel,internal temperature probe, and nitrogen purge is charged with 1000 g ofthe dextran and 9.00 L of de-ionized water. The mixture is stirred atambient temperature to dissolve the dextran and then cooled to 10 to 15°C. To the cooled dextran solution is added over a period of an hour,while keeping the reaction temperature below 25° C., a solution of 1000g of sodium periodate dissolved in 9.00 L of de-ionized water. Once allthe sodium periodate solution has been added, the mixture is stirred at20 to 25° C. for 4 more hours. The reaction mixture is then cooled to 0°C. and filtered to clarify. Calcium chloride (500 g) is added to thefiltrate, and the mixture is stirred at ambient temperature for 30 minand then filtered. Potassium iodide (400 g) is added to the filtrate,and the mixture is stirred at ambient temperature for 30 min. A 3-Lportion of the resulting red solution is added to 9.0 L of acetone overa period of 10 to 15 min with vigorous stirring by a mechanical stirrerduring the addition. After a few more minutes of stirring, theagglomerated product is separated from the supernatant liquid. Theremaining red solution obtained by addition of potassium iodide to thesecond filtrate is treated in the same manner as above. The combinedagglomerated product is broken up into pieces, combined with 2 L ofmethanol in a large stainless steel blender, and blended until the solidbecomes granular. The granular solid is recovered by filtration anddried under vacuum with a nitrogen purge. The granular solid is thenhammer milled to a fine powder. A 20-L reactor is charged with 10.8 L ofde-ionized water and 7.2 L of methanol, and the mixture is cooled to 0°C. The granular solid formed by the previous step is added to thereactor and the slurry is stirred vigorously for one hour. Stirring isdiscontinued, and the solid is allowed to settle to the bottom of thereactor. The supernatant liquid is decanted by vacuum, 15 L of methanolis added to the reactor, and the slurry is stirred for 30 to 45 minwhile cooling to 0° C. The slurry is filtered in portions, and therecovered solids are washed with methanol, combined, and dried undervacuum with a nitrogen purge to give about 600 g of the oxidizeddextran, which is referred to herein as D10-50.

The degree of oxidation of the product is determined by proton

NMR to be about 50% (equivalent weight per aldehyde group=146). In theNMR method, the integrals for two ranges of peaks are determined,specifically, —O₂CHx— at about 6.2 parts per million (ppm) to about 4.15ppm (minus the HOD peak) and —OCHx— at about 4.15 ppm to about 2.8 ppm(minus any methanol peak if present). The calculation of oxidation levelis based on the calculated ratio (R) for these areas, specifically,R=(OCH)/(O₂CH).

Preparation of Eight-Arm PEG 10K Octaamine (P8-10-1):

Eight-arm PEG 10K octaamine (M_(n)=10 kDa) is synthesized using thetwo-step procedure described by Chenault in co-pending and commonlyowned U.S. Patent Application Publication No. 2007/0249870. In the firststep, the 8-arm PEG 10K chloride is made by reaction of thionyl chloridewith the 8-arm PEG 10K octaalcohol. In the second step, the 8-arm PEG10K chloride is reacted with aqueous ammonia to yield the 8-arm PEG 10Koctaamine. A typical procedure is described here.

The 8-arm PEG 10K octaalcohol (M_(n)=10000; NOF SunBright HGEO-10000),(100 g in a 500-mL round-bottom flask) is dried either by heating withstirring at 85° C. under vacuum (0.06 mm of mercury (8.0 Pa)) for 4hours or by azeotropic distillation with 50 g of toluene under reducedpressure (2 kPa) with a pot temperature of 60° C. The 8-arm PEG 10Koctaalcohol is allowed to cool to room temperature and thionyl chloride(35 mL, 0.48 mol) is added to the flask, which is equipped with a refluxcondenser, and the mixture is heated at 85° C. with stirring under ablanket of nitrogen for 24 hours. Excess thionyl chloride is removed byrotary evaporation (bath temp 40° C.). Two successive 50-mL portions oftoluene are added and evaporated under reduced pressure (2 kPa, bathtemperature 60° C.) to complete the removal of thionyl chloride. ProtonNMR results from one synthesis are:

¹H NMR (500 MHz, DMSO-d6) δ 3.71-3.69 (m, 16H), 3.67-3.65 (m, 16H), 3.50(s, ˜800H).

The 8-arm PEG 10K octachloride (100 g) is dissolved in 640 mL ofconcentrated aqueous ammonia (28 wt %) and heated in a pressure vesselat 60° C. for 48 hours. The solution is sparged for 1-2 hours with drynitrogen to drive off 50 to 70 g of ammonia. The solution is then passedthrough a column (500 mL bed volume) of strongly basic anion exchangeresin (Purolite® A-860, The Purolite Co., Bala-Cynwyd, Pa.) in thehydroxide form. The eluant is collected and three 250-mL portions ofde-ionized water are passed through the column and also collected. Theaqueous solutions are combined, concentrated under reduced pressure (2kPa, bath temperature 60° C.) to about 200 g, frozen in portions andlyophilized to give the 8-arm PEG 10K octaamine, referred to herein asP8-10-1, as a colorless waxy solid.

Preparation of 8-Arm PEG 10K Hexadecaamine (P8-10-2):

An 8-arm PEG 10K hexadecaamine, referred to herein as “P8-10-2”, havingtwo primary amine groups at the end of the arms, was prepared using atwo step procedure, as described by Arthur in WO 2008/066787, in which8-arm PEG 10K was reacted with methanesulfonyl chloride indichloromethane in the presence of triethylamine to produce 8-arm PEG10K mesylate, which was subsequently reacted withtris(2-aminoethyl)amine to give the 8-arm PEG 10K hexadecaamine. Atypical synthesis is described here.

To a solution of 10 g of 8-arm PEG 10K (M_(n)=10,000; NOF, Tokyo, Japan)in 50 mL of dichloromethane stirred under nitrogen and cooled to 0° C.is added 2.2 mL of triethylamine, followed by 1.2 mL of methanesulfonylchloride. The mixture is allowed to warm to room temperature and isstirred overnight. The reaction mixture is transferred to a separatoryfunnel and washed gently three times with 15 mL portions of 1 Mpotassium dihydrogen phosphate, followed by 15 mL of 1 M potassiumcarbonate, and then 15 mL of water. The dichloromethane layer is driedover magnesium sulfate, filtered, and concentrated by rotary evaporationto afford 11.17 g of 8-arm PEG 10K mesylate.

A mixture of 10 g of 8-arm PEG 10K mesylate and 45 mL oftris(2-aminoethyl)amine dissolved in 45 mL of water is stirred at roomtemperature for 24 hours. The reaction mixture is diluted with 45 mL of5% (w/w) aqueous sodium bicarbonate and extracted with a total of 500 mLof dichloromethane divided in 3 portions. The dichloromethane solutionis dried over sodium sulfate, and concentrated by rotary evaporation to20 to 25 g. Ether (100 mL) is added to the concentrated dichloromethanesolution with vigorous stirring, and the mixture is cooled to 0° C.,causing a waxy solid to separate from solution. The solvent is decantedfrom the waxy solid, and the waxy solid is dried under vacuum to givethe 8-arm PEG 10K hexadecaamine (P8-10-2).

EXAMPLES 1-15 In-Vitro Burst Testing of a Sealed Scalpel Incision inSwine Uterine Horn

The purpose of these Examples was to demonstrate the burst strength of aseal made with various hydrogels of an incision made in swine uterinehorn.

A syringe pump system was used to measure the burst strength of a sealof an incision made in a section of swine uterine horn. The syringe pump(Model No. 22, Harvard Apparatus, Holliston, Mass.) was modified to beequipped with two 30 mL syringes, which were connected together througha “Y” junction. Water was pumped through a single piece of Tygon® R-36tubing (0.6 cm diameter) and through a pressure gauge (Model PDG 5000L,Omega Engineering, Stamford, Conn.). An approximately 12.5 cm section ofclean swine uterine horn, obtained from a local abattoir, was fitted onone end with a metal plug with a feed line fitting for water feed fromthe syringe pump and on the other end with a metal plug with a threadedhole which could be sealed with a machine screw. The plugs were held inplace with nylon ties around the outside of the intestine. An incisionwas made through the uterine horn wall into the interior by puncturingwith a Bard Parker™ surgical blade handle 5 (obtained from BD SurgicalProducts, Franklin Lakes, N.J.), fitted with a #15 surgical blade. Theincision on the outside of the uterine horn was wider than the scalpelblade (typically 4-5 mm) while the hole through the inside wall wasabout 3 mm (about equal to the blade). This size incision mimics thedistance between the interrupted sutures if an intestine were to be cutand later sutured. The uterine horn was filled with water containing apurple dye via the syringe pump until water began to leak from the openhole in the end plug and also from the scalpel puncture in the uterinehorn wall. The pump was then turned off and the end plug was sealed withthe machine screw. The scalpel incision site was blotted dry using apaper towel.

The aldehyde-functionalized polysaccharide and multi-arm PEG aminesolutions, as shown in Table 1, were prepared in water with shakingovernight at 37° C. and 175 rpm. The two solutions were applied to theincision using a double barrel syringe (Mixpac Systems AG (Rotkreuz,Switzerland) fitted with a 16 or a 12 step static mixer (Mixpac SystemsAG). After the application, the adhesive was allowed to cure at roomtemperature for no longer than 2 min. Burst pressure testing, alsoreferred to herein as leak pressure testing, was done by pressurizingthe sealed intestine with water from the syringe pump at a flow rate of11 mL/min until the bioadhesive seal began to leak, at which point thepressure was recorded. Adhesive failure was attributed when the waterleaked under the seal between the hydrogel and the tissue surface.Cohesive failure was attributed when the water penetrated and leakedthrough the hydrogel itself. The results of the burst testing aresummarized in Table 1.

TABLE 1 Burst Pressure Testing Results Aldehyde- FunctionalizedPolysaccharide Multi-Arm PEG Ave Burst Example Solution Amine SolutionPressure, psi 1 AFD-15-90 P8-10-1/P8-10-2 1.2 40 wt % (1:2 w/w) (8.3kPa)  20 wt % 2 AFD-15-90 P8-10-1/P8-10-2 2.4 40 wt % (1:2 w/w) (16 kPa)30 wt % 3 AFD-15-90 P8-10-1/P8-10-2 2.8 25 wt % (1:2 w/w) (19 kPa) 20 wt% 4 AFD-15-90 P8-10-1/P8-10-2 1.3 25 wt % (1:2 w/w) (9.0 kPa)  10 wt % 5AFD-7-86 P8-10-1 4.2 30 wt % 50 wt % (29 kPa) 6 AFD-7-86 P8-10-2 1.1 30wt % 20 wt % (7.6 kPa)  7 AFD-7-86 P8-10-1/P8-10-2 2.7 30 wt % (9:1 w/w)(19 kPa) 30 wt % 8 AFD-9-120 P8-10-1 3.0 25 wt % 30 wt % (21 kPa) 9AFD-9-120 P8-10-1/P8-10-2 2.5 25 wt % (1:2 w/w) (18 kPa) 25 wt % 10AFD-13-64 P8-10-1 2.5 25 wt % 30 wt % (18 kPa) 11 AFD-13-64P8-10-1/P8-10-2 4.2 25 wt % (1:2 w/w) (30 kPa) 25 wt % 12 AFI-12-49P8-10-1/P8-10-2 2.6 40 wt % (9:1 w/w) (18 kPa) 30 wt % 13 AFI-12-49P8-10-1/P8-10-2 2.8 25 wt % (9:1 w/w) (19 kPa) 30 wt % 14 AFI-12-49P8-10-1/P8-10-2 4.7 40 wt % (1:1 w/w) (32 kPa) 30 wt % 15 AFI-12-49P8-10-1/P8-10-2 4.2 40 wt % (1:2 w/w) (29 kPa) 30 wt %

The results shown in Table 1 demonstrate that hydrogels formed byreacting an aldehyde-functionalized polysaccharide containing pendantsingle aldehyde groups with a mixture of an eight-arm branched endpolyethylene glycol amine having two primary amine groups at the end ofthe polymer arms (i.e. P8-10-2) and an eight-arm polyethylene glycolamine (P8-10-1) having one primary amine group at the end of the polymerarms, adhered well to and sealed biological tissue.

EXAMPLES 16-26 In Vitro Biocompatibility Testing—Cytotoxicity

The purpose of these Examples was to demonstrate the safety of hydrogelsresulting from the reaction of an aldehyde-functionalized polysaccharidewith a multi-arm PEG amine in an in vitro test.

The testing was done using NIH3T3 mouse fibroblast cell culturesaccording to ISO10993-5:1999. The NIH3T3 mouse fibroblast cells wereobtained from the American Type Culture Collection (ATCC; Manassas, Va.)and were grown in Dulbecco's modified essential medium (DMEM),supplemented with 10% fetal calf serum.

NIH3T3 mouse fibroblast cell cultures were challenged with hydrogelsmade by combining equal volumes of an aqueous solution of analdehyde-functionalized polysaccharide and an aqueous solution ofmulti-arm PEG amine, as shown in Table 2. The aqueous solutions wereprepared and mixed to form hydrogels as described in Examples 1-15. Eachhydrogel was placed in the bottom of a well in a polystyrene cultureplate such that about ¼ of the well bottoms were covered. The wells werethen sterilized under UV light and seeded with 50,000-100,000 NIH3T3cells.

The cells grew normally confluent and coated the well bottom, growing upto the edges of the hydrogels; however, they did not overgrow thehydrogels. These results, summarized in Table 2, demonstrate a lack ofcytotoxicity of the hydrogels, as well as the lack of adhesion of cellcultures to the hydrogels.

TABLE 2 Cytotoxicity Results Aldehyde- Functionalized PolysaccharideMulti-Arm PEG Example Solution amine solution Cytotoxicity 16 AFI-12-49P8-10-1/P8-10-2 nontoxic 25 wt % (9:1 w/w) 30 wt % 17 AFI-12-49P8-10-1/P8-10-2 nontoxic 40 wt % (1:1 w/w) 30 wt % 18 AFI-12-49P8-10-1/P8-10-2 nontoxic 40 wt % (1:2 w/w) 25 wt % 19 AFD-9-120 P8-10-1nontoxic 25 wt % 30 wt % 20 AFD-9-120 P8-10-1/P8-10-2 nontoxic 25 wt %(1:2 w/w) 25 wt % 21 AFD-13-64 P8-10-1 nontoxic 25 wt % 30 wt % 22AFD-13-64 P8-10-1/P8-10-2 nontoxic 25 wt % (1:2 w/w) 25 wt % 23AFD-15-90 P8-10-1/P8-10-2 nontoxic 25 wt % (1:2 w/w) 20 wt % 25AFD-15-90 P8-10-1/P8-10-2 nontoxic 40 wt % (1:2 w/w) 20 wt % 26AFD-15-90 P8-10-1/P8-10-2 nontoxic 40 wt % (1:2 w/w) 30 wt %

EXAMPLES 27-37 In Vitro Degradation of Hydrogels

The purpose of these Examples was to demonstrate that hydrogels formedby the reaction of an aldehyde-functionalized polysaccharide with amulti-arm PEG amine are hydrolyzed readily in an in vitro test.

The hydrogel samples were prepared by mixing equal volumes of an aqueoussolution of an aldehyde-functionalized polysaccharide and an aqueoussolution of a multi-arm PEG amine, as shown in Table 3. The aqueoussolutions were prepared and mixed to form hydrogels as described inExamples 1-15. After the hydrogels cured, the samples were weighed andplaced inside jars containing PBS (phosphate buffered saline) at pH 7.4.The jars were placed inside a temperature-controlled shaker set at 80rpm and 37° C. The samples were removed from the jars at various times,blotted to remove excess solution, and weighed. Then, the samples werereturned to the jars.

The results are summarized in Table 3. The percent swell reported in thetable is the weight of the hydrogel measured during the course of thestudy divided by the initial weight of the hydrogel, multiplied by 100.

TABLE 3 In Vitro Degradation of Hydrogels Aldehyde- FunctionalizedPolysaccharide Multi-Arm PEG Time Example Solution Amine Solution(hours) % Swell 27 AFD-15-90 P8-10-1/P8-10-2 0 100 40 wt % (1:2 w/w) 6332 30 wt % 24 285 54 259 96 243 192 218 216 207 264 203 384 191 456 184528 176 28 AFD-15-90 P8-10-1/P8-10-2 0 100 40 wt % (1:2 w/w) 6 150 20 wt% 24 102 54 87 96 74 192 64 216 59 264 58 384 46 456 51 528 49 29AFD-15-90 P8-10-1/P8-10-2 0 100 25 wt % (1:2 w/w) 6 214 20 wt % 24 17654 152 96 129 192 125 216 125 264 112 384 109 456 107 528 107 30AFD-7-86 P8-10-1 6 79 40 wt % 50 wt % 24 0 31 AFI-12-49 P8-10-1/P8-10-26 0 40 wt % (9:1 w/w) 75 0 30 wt % 32 AFD-13-64 P8-10-1 3 20 20 wt % 30wt % 6 0 33 AFD-13-64 P8-10-1/P8-10-2 3 58 30 wt % (9:1 w/w) 6 0 30 wt %34 AFD-13-64 P8-10-1/P8-10-2 6 366 30 wt % (1:1 w/w) 48 223 30 wt % 31259 35 AFI-12-49 P8-10-1/P8-10-2 6 0 40 wt % (9:1 w/w) 30 wt % 36AFI-12-49 P8-10-1/P8-10-2 6 0 40 wt % (1:1 w/w) 75 0 30 wt % 37AFI-12-49 P8-10-1/P8-10-2 6 116 40 wt % (1:2 w/w) 75 20 25 wt %

The results in Table 3 demonstrate that hydrogels formed by the reactionof an aldehyde-functionalized polysaccharide with a multi-arm PEG amineare hydrolyzed readily in an in vitro test. By using different amountsof the components in terms of wt % and/or by altering the amount offuntionalization of either amine on the PEG amines or aldehyde on thealdehyde-functionalized polysaccharide, the time for degradation can betuned from a few hours to many days. The hydrolysis results suggest thatthe hydrogels disclosed herein should degrade readily in vivo.

EXAMPLES 38 and 39 In Vitro Degradation of Hydrogels Formed UsingDialdehyde-Functionalized Dextrans

The purpose of these Examples was to examine the in vitro degradation ofhydrogels formed by the reaction of dialdehyde-functionalized dextranswith a multi-arm PEG amine.

An aqueous solution containing dialdehyde-functionalized dextranDAFD-10-16 (Example 38) or DAFD-10-Ester Linked (Example 39) at aconcentration of 20 wt % was mixed with an aqueous solution containingmulti-arm PEG amine P8-10-1 (25 wt %) to form a hydrogel, as describedin Examples 1-15. The degradation of the hydrogels was determined usingthe method described in Examples 27-37. The results are summarized inTable 4.

TABLE 4 In Vitro Degradation of Hydrogels Aldehyde- FunctionalizedPolysaccharide Multi-Arm PEG Time Example Solution Amine Solution(hours) % Swell 38 DAFD-10-16 P8-10-1 0 100 20 wt % 25 wt % 1 249 2 2584 245 6 244 8 243 22 244 26 247 39 DAFD-Ester P8-10-1 0 100 Linked 25 wt% 1 236 20 wt % 2 45.3 4 3.6

These results demonstrate that dialdehyde functionalized dextrans formhydrogels gels with the multi-arm PEG amine P-8-10-1 and that hydrogelswith a wide range of degradation rates in an aqueous environment can beobtained. Specifically, long lived hydrogels can be formed usingaldehyde-functionalized dextran having pendant dialdehyde groups wherethe linkage to the dextran backbone is chemically stable as in the etherlinked dialdehyde-functionalized dextran (Example 38). Much fasterdegrading hydrogels can also be prepared by usingaldehyde-functionalized dextran having pendant dialdehyde groups wherethe linkage of the the pendant dialdehyde groups is potentiallyhydrolytically unstable, such as an ester linkage (Example 39).

EXAMPLES 40-44 Stability of Aldehyde-Functionalized Dextrans in AqueousSolution—Viscosity Measurements

The purpose of these Examples was to demonstrate the higher stability ofaldehyde-functionalized dextrans in aqueous solution compared tooxidized dextran D10-50 using viscosity measurements.

Aqueous solutions of oxidized dextran (25 wt %) and variousaldehyde-functionalized dextrans (25 wt %), as shown in Table 5, wereheated at 45° C. for 12 days. The viscosity of the aqueous solutions wasmeasured at 30° C. at various time points. The results are summarized inTable 5.

TABLE 5 Viscosity of Aqueous Solutions of Oxidized dextran andAldehyde-Functionalized Dextran Viscosity Viscosity Viscosity ViscosityDecrease (cP) (cP) (cP) after 12 Example Dextran Day 0 Day 6 Day 12 days40 AFD-13- 17.37 17.35 17.94 0% 64 41 AFD-13- 12.6 13.0 11.0 11% 46 42AFD-19- 15.7 17.6 15.1 4% 64 43 AFD-15- 14.3 14.3 12.7 11% 90 44, D10-5020 18 15.6 22% Comparative

The results in Table 5 show that the aqueous solutions containing thealdehyde-functionalized dextrans having pendant aldehyde groups had adecrease in viscosity after 12 days ranging from 0% to 11%, whereas theviscosity of the aqueous solution containing the oxidized dextrandecreased by 22% over the same time period. These results suggest thatthe aldehyde-functionalized dextrans having pendant aldehyde groups aremore stable in aqueous solution than the oxidized dextran.

EXAMPLES 45-48 Stability of Aldehyde-Functionalized Dextrans in AqueousSolution—Rheometry Measurements

The purpose of these Examples was to demonstrate the stability ofaldehyde-functionalized dextrans in aqueous solution. Oscillating diskrheometry of the hydrogels resulting from the reaction of thealdehyde-functionalized dextrans with multi-arm polyether amines wasused as a measure of the stability of the aldehyde-functionalizeddextran solutions.

Double barrel syringes containing an aqueous solution of variousaldehyde-functionalized polysaccharides in one barrel and an aqueoussolution of a multi-arm PEG amine in the other barrel, as shown in Table6, were prepared in duplicate. Two other double barrel syringes werefiled with an aqueous solution of oxidized dextran D10-50 in one barreland an aqueous solution of P8-10-1 (20 wt %) in the other barrel. Theaqueous solutions contained in the double barrel syringes were expressedthrough a static mixing tip onto the sample platform of a Model APA2000rheometer (Alpha Technologies, Akron, Ohio), and the storage modulus(G′) of the mixture was measured and taken as the value on Day zero. Thevalue of G′ at 60 seconds was taken as a measure of speed of gelation.

One group of the syringes was stored at 25° C. for various periods oftime and the second group of syringes was heated at 40° C. for variousperiods of time (as shown in Table 6) to provide thermally aged aqueoussolutions of the aldehyde-functionalized polysaccharides. The storagemodulus of the mixed solutions was measured as described above atvarious times. The results are shown in Table 6, expressed as thepercent of the Day zero G′.

TABLE 6 Rheometry Results of Hydrogel Formation Alehyde- Percent Percentof Functionalized Multi-Arm of Day 0 Day 0 G′ Exam- Polysaccharide PEGG′ (60 sec) (60 sec) ple Solution Amine Solution 25° C. 40° C. 45AFD-15-90 P8-10-1 94% 56% (19 wt %) (20 wt %) (Day 20) (Day 20) 46AFD-16-92 P8-10-1 96% 58% (20 wt %) (20 wt %) (Day 26) (Day 26) 47AFI-12-49 P8-10-2/P8-10- 94% 81% (25 wt %) 1 (2:1) 20 wt % (Day 26 (Day26) 48, D10-50 P8-10-1 75% Did not gel Compara- (25 wt %) (20 wt %) (Day30) (Day 30) tive

The results in Table 6 suggest that the aldehyde-functionalizedpolysaccharides are more stable in aqueous solution than oxidizeddextran.

EXAMPLE 49 Thermal Stability of AFD-13-64 in Aqueous Solution UsingRheometry Measurements

The purpose of this Example was to demonstrate the thermal stability ofaldehyde-functionalized dextran AFD-13-64 in aqueous solution usingoscillating disk rheometry.

Two double barrel syringes, each containing an aqueous solution ofAFD-13-64 (20 wt %) in one barrel and an aqueous solution of P8-10-1 (20wt %) in the other barrel were prepared. One syringe was heated at 40°C. for 19 days; the other syringe was stored at 4° C. for the sameperiod of time. Then, the storage modulus of the mixtures resulting fromeach syringe was measured using a Model APA2000 rheometer (AlphaTechnologies, Akron, Ohio). The storage modulus obtained for the mixtureresulting from the syringe that was stored at 40° C. for 19 days wasessentially identical to that obtained from the mixture resulting fromthe syringe that was stored at 4° C. for the same period of time,suggesting that the aldehyde-functionalized dextran AFD-13-64 has goodthermal stability.

EXAMPLE 50, COMPARATIVE Aldehyde-Functionalized CarboxymethyldextranContaining Pendant Aldehyde Groups Attached by an Amide Linkage

The purpose of this Example was to demonstrate that analdehyde-functionalized polysaccharide containing pendant aldehydegroups that are attached to the polysaccharide by an amide linkage isnot as stable in aqueous solution as an aldehyde-functionalizedpolysaccharide containing pendant aldehyde groups that are attached tothe polysaccharide by an ether linkage.

Preparation of 11 kDa Carboxymethyldextran with Degree ofCarboxymethylation of 1.1 (CMDX-11-1.1):

Dextran having an average molecular weight of 8.5-11 kDa (Sigma) wasdissolved in 123.75 mL of 6 N NaOH at 0° C. To this cold solution wasadded 30.75 g of chloroacetic acid (Aldrich). This reaction mixture washeated to 60° C. for 20 min, then cooled, and neutralized to pH 7.0 withconcentrated HCl. The product was precipitated by adding the neutralizedsolution dropwise to 1.0 L of methanol. The solids were collected byfiltration and re-precipitated from methanol. The entire procedure wasthen repeated three times to raise the degree of substitution to thedesired level. After the final repeat, the product was further purifiedby ultrafiltration. The solution was diafiltered using a MilliporePellicon II TFF system. A total of 6 volumes of permeate was collectedwhile continuously adding water to maintain a constant retentate volume.The retentate was then collected and lyophilized to give 14.25 g of afluffy white solid. The degree of carboxymethylation was determined bythe method of Ho et al. (Anal. Chem. 52:916, 1980) to be 1.1. Thiscarboxymethyldextran product is referred to herein as CMDX-11-1.1. ¹HNMR δ 4.9-5.2 mult. (1H), 3.4-4.4 mult (9.5H).

Preparation of Aldehyde-Functionalized Carboxymethyldextran ContainingPendant Aldehyde Groups Attached by an Amide Linkage:

To a 3 liter, 3 necked flask was added 13.7 g of CMDX-60-1.7 and 916.6mL of a 1:1 solution of teramethylethylene diamine buffer anddimethylformamide (DMF). A clear solution having a of pH 4.94 wasformed. The pH was adjusted to 4.7 by addition of 1.0 M HCl. To thesolution was added 47.74 g of1-ethyle-3(3-dimethylaminopropyl)carbodiimide (Sigma) followed by 28.87g of n-hydroxysuccinimide (Aldrich). The pH of the solution wasreadjusted to 4.7 and stirred for 2 hours. Then, 4-aminobutyraldehdyediethyl acetal (53.88 g) was added in portions over 3.5 hours so thatthe pH did not rise above 4.7. Toward the end of the reaction, the pHwas allowed to rise to 6.25 and then the reaction mixture was stirredovernight. The reaction mixture was transferred to a glass jar, dilutedwith water and filtered on a Millipore Pellicon II ultrafiltrationsystem with 1 kDa cutoff filters. A total of 5 volumes were collected aspermeate while continuously recycling and adding fresh water to replacethe permeate. The filtered solution was lyophilized to give a whitesolid product. The above procedure was then repeated starting with thewhite solid product to increase the loading of pendant acetal groups.The final yield was 20.3 g.

The acetal groups were removed by dissolving the solid in 400 mL ofwater and treating with 1.0 M HCl at pH 2.5 overnight. Afterneutralizing with NaOH, the solution was filtered on the MilliporePellicon II ultrafiltration system as previously described andlyophilized to give 16.45 g of white solid. The molecular weight wasdetermined by size exclusion chromatography to be 49 kDa. The degree ofaldehyde substitution per ring was determined by NMR of the hydrolizedsample to be 0.37. In the NMR method, the integrals for two ranges ofpeaks are determined, specifically, —O₂CHx— at about 6.2 parts permillion (ppm) to about 4.15 ppm (minus the HOD peak) and —OCHx— at about4.15 ppm to about 2.8 ppm (minus any methanol peak if present). Thecalculation of oxidation level is based on the calculated ratio (R) forthese areas, specifically, R=(OCH)/(O₂CH).

Instability of the Aldehyde-Functionalized Carboxymethyldextran inAqueous Solution:

An aqueous solution of aldehyde-functionalized carboxymethyldextrancontaining pendant aldehyde groups attached by an amide linkage was madeby dissolving 5.1 g of the solid product obtained as described above in15.3 g of autoclaved water. The mixture was shaken at 190 rpm in anincubator at 37° C. for 1 hour. The resulting solution was filteredthrough a 5.0 pm membrane and two 5 mL samples of the solution wereplaced in 55° C. incubator. After 19 hours both samples had formed ambergels which exhibited no flow.

What is claimed is:
 1. A method for applying a coating to an anatomicalsite on tissue of a living organism comprising the steps of applying tothe site a) at least one aldehyde-functionalized polysaccharidecontaining pendant aldehyde groups, said aldehyde-functionalizedpolysaccharide having a weight-average molecular weight of about 1,000to about 1,000,000 Daltons and a degree of aldehyde substitution ofabout 10% to about 200%; followed by b) at least one water-dispersible,multi-arm amine wherein at least three of the arms are terminated by atleast one primary amine group, said multi-arm amine having anumber-average molecular weight of about 450 to about 200,000 Daltons,or (b) followed by (a), or premixing (a) and (b) and applying theresulting mixture to the site before the resulting mixture completelycures.
 2. The method according to claim 1 wherein thealdehyde-functionalized polysaccharide is selected from the groupconsisting of aldehyde-functionalized derivatives of: dextran,carboxymethyldextran, starch, agar, cellulose, hydroxyethylcellulose,carboxymethylcellulose, pullulan, inulin, levan, and hyaluronic acid. 3.The method according to claim 1 wherein the water-dispersible, multi-armamine is selected from the group consisting of water-dispersiblemulti-arm polyether amines, amino-terminated dendritic polyamidoamines,and multi-arm branched end amines.